1. Field of the Invention
The present invention relates generally to the fields of plant biotechnology and molecular genetics of eucaryotic gene expression and translation. More specifically, the present invention relates to novel cap-independent translation sequences derived from barley yellow dwarf virus.
2. Description of the Related Art
Translation is the step in gene expression at which proteins are synthesized. The genetic code is decoded from a messenger RNA (mRNA) sequence to protein sequence. All genes that encode proteins must be translated. mRNAs of eukaryotes (plants, animals and fungi) are transcribed (synthesis of mRNA from DNA) by RNA polymerase II. All polymerase II transcripts, except histone genes, are modified post-transcriptionally to contain a m.sup.7 G cap structure at the 5' end (beginning) and a polyadenylate tract (poly(A) tail) at the 3' end (end). These modifications are required for translation of the mRNA.
Models for translation initiation in eukaryotes call for ribosome recognition of the 5' cap of mRNA, mediated by initiation factors, followed by 5' to 3' scanning until the first AUG is reached at which protein synthesis ensues.sup.1,2. However, the 3' untranslated region communicates with the 5' end of an mRNA in regulating initiation of translation. Cis-acting signals in the 3' untranslated regions of certain mRNAs can be recognized by specific regulatory proteins that inhibit translation initiation.sup.3. Poly(A) tails.sup.4,5 or functionally equivalent domains in some plant viruses.sup.6, stimulate initiation of translation. The 5' cap and poly(A) tail act synergistically to stimulate initiation in vivo.sup.5 but cannot substitute for each other. The 5' cap strongly stimulates initiation in reticulocyte lysates and wheat germ, but stimulation by poly(A) is minimal in either system.sup.5,7. In addition to stimulating translation initiation, both components confer mRNA stability in vivo.sup.8.
Sequences in the 5' UTRs of some viral RNAs can substitute for a cap.sup.9-11. For example, the Internal Ribosome Entry Site (IRES) of picornaviruses.sup.10,12 facilitates 40S ribosomal subunit binding far from the 5' end of the mRNA (which lacks a cap) in the absence of translation factors (eIF-4F) that are necessary for cap-dependent translation initiation. Like the 5' cap, these sequences are located 5' of the AUG codon at which protein synthesis begins.
A sequence (3' translation enhancer, 3'TE) located between bases 4513 and 5009 of the 5677 nucleotide genomic RNA of barley yellow dwarf virus, PAV serotype (PAV) facilitates efficient translation when located in the 3' untranslated regions of uncapped viral or heterologous mRNAs in wheat germ translation extracts (WGE).sup.13. These constructs also contained the viral 5' untranslated region. A similar phenomenon has been observed with the 1200 nucleotide satellite tobacco necrosis virus (STNV) RNA in wheat germ translation extracts .sup.14,15. In none of these cases has the 3' untranslated region been shown to function in vivo.
Sequences in the 5'UTR and 3'UTR of satellite tobacco necrosis virus also confer efficient translation on uncapped mRNAs in wheat germ translation extracts (Danthinne et al., 1993; Timmer et al., 1993). The 5'UTR of tobacco etch virus (TEV) confers efficient translation of uncapped mRNAs in vitro and in vivo and translation is stimulated a further 15-fold in plant cells when the mRNA containing the tobacco etch virus 5'UTR is capped (Carrington and Freed, 1990). The 5'UTRs of alfalfa mosaic virus (AIMV) and tobacco mosaic virus (.OMEGA. sequence of tobacco mosaic virus) confer efficient translation of capped mRNAs (Sleat and Wilson, 1992). Both are used in vectors for high level gene expression in transgenic plants. The barley yellow dwarf virus-PAV translation enhancing sequences show no apparent sequence similarity to any of the above sequences.
The satellite tobacco necrosis virus and tobacco etch virus sequences do not seem to confer as much stimulation of translation of uncapped RNAs in vitro. The activity of satellite tobacco necrosis virus sequences in cells or plants is unknown. The alfalfa mosaic virus and tobacco mosaic virus sequences differ from the barley yellow dwarf virus-PAV sequences in that they do not confer efficient translation of uncapped mRNAs in vivo. The tobacco etch virus sequence does not fully substitute for the presence of a 5' cap.
Almost all eukaryotic cellular mRNAs contain a 5' m.sup.7 G(5')ppp(5')N cap structure that is required for efficient initiation of translation. According to the ribosome scanning model of eukaryotic translation initiation, initiation factor eIF4F specifically recognizes the 5' cap structure and, with other initiation factors such as eIF3, recruits the 43S ribosomal subunit initiation complex that then scans 5' to 3' along the mRNA. When the first (or second in the case of leaky scanning) AUG codon is reached, the 60S ribosomal subunit joins and peptide elongation ensues.
Although the ribosome scanning model explains the mechanisms of various translational regulatory elements in the 5' UTRs of mRNAs, numerous examples exist of translational control elements in the 3'UTRs of mRNAs. The 3' poly(A) tail, found on most eukaryotic cellular mRNAs, stimulates translation initiation and stabilizes mRNA. The 5' cap and poly(A) tail act synergistically to stimulate initiation in vivo and in a yeast in vitro translation system. Viral RNAs that lack a poly(A) tail, sequences in the 3'UTR, such as the pseudoknot-rich domain of tobacco mosaic virus (TMV) appear to functionally substitute for a poly(A) tail to stimulate translation in conjunction with the 5' cap. Other cis-acting elements in 3' UTRs control translation initiation either by modulating poly(A) tail length during embryo development, or via binding of a specific regulatory protein that inhibits initiation. In all these examples, the mRNA must have a 5' cap for the 3' element to function. Thus, in previously described mRNAs, cap recognition is likely an essential component in the communication between 3' and 5' ends.
Mammalian eIF4F complex is comprised of eIF4E (the cap-binding subunit), eIF4G (p220), and the loosely-associated eIF4A (helicase). Plant cells contain two forms of the 4F complex: eIF4F, consisting of 26 kDa and 220 kDa subunits (homologous to eIF4E and eIF4G, respectively), and eIFiso4F, consisting of 28 kDa and 86 kDa subunits. eIF4A probably has the same function in plants but does not co-purify with eIF4F, so it is not considered a subunit of this complex. In defined cell-free systems, capped mRNAs have a reduced requirement for eIF4F compared to uncapped mRNAs.
Many viral mRNAs lack a 5' cap or a poly(A) tail, or both. They have evolved ways of ensuring efficient translation of their genes from uncapped mRNAs, often at the expense of the host cell. The most well-documented example is the translation of picornavirus RNAs. Picornaviral RNAs lack a 5' cap structure and have an extremely long, highly structured 5' untranslated region, including many AUG codons upstream of the start codon of the main open reading frame. Rather than scanning from the 5' end, ribosomes bind internally in this long leader at the internal ribosomal entry site just upstream of the start codon, in a cap-independent manner. Although IRES-mediated cap-independent translation does not comform to the rule of 5' cap-eIF4E recognition, it still employs the other canonical initiation factors including the eIF4G subunit of eIF4F, and follows the scanning concept of ribosome binding the 5' UTR followed by scanning in a 3' direction until the appropriate start codon is reached for initiation of translation.
The prior art is deficient in the lack of effective means of stimulating high level expression of proteins from uncapped mRNAs in vivo. The present invention fulfills this longstanding need and desire in the art.